Nucleotide sequences coding for the bovine β3 -adrenergic receptor (ARβ3) and their applications

ABSTRACT

Nucleotidic sequences coding for the bovine adrenergic-β 3  receiver (RAβ 3 ), utilization of said sequences as probes and for the expression of peptides and/or fragments thereof having an activity of bovine RAβ 3 , vectors useful for said expression, as well as cellular hosts containing said vector. Polyclonal and monoclonal antibodies raised against said peptides and usable, particularly for the detection of bovine adrenergic-β 3  receivers, as well as method for screening substances, with agonist or antagonist action in relation to peptides having an adrenergic-β 3  receiver activity and kits for the detection of the affinity degrees of different substances for said peptides having an adrenergic-β 3  receiver activity. Transgenic and recombinant mice containing said nucleotidic sequence.

This application was filed as application PCT/FR94/00447 on Apr. 21,1994, and it entered the national stage on Feb. 21, 1995.

BACKGROUND OF INVENTION

1. Field of the Invention

The present invention relates to nucleotide sequences coding for thebovine β₃ -adrenergic receptor (ARβ₃), to the use of the said sequencesas probes and for the expression of peptides and/or of fragments of thelatter having bovine ARβ₃ activity, to the vector which is useful forthe said expression and also to host cells containing the said vector.

The present invention also relates to a method for the screening ofsubstances possessing an agonist or antagonist action with respect topeptides of bovine origin having β₃ -adrenergic receptor activity.

2. Description of the Related Art

Catecholamines such as adrenaline and noradrenaline, synthetic agonistsof these catecholamines which mimic their biological functions andantagonists which block these biological functions are known to exerttheir effects by binding to specific recognition sites (membranereceptors) located on the cell membranes.

Two main classes of adrenergic receptors have been defined, α-adrenergicreceptors and β-adrenergic receptors.

In the set of these two classes, five subtypes of catecholaminereceptors are now distinguished (α₁ -, α₂ -, β₁ -, β₂ - and β₃ -AR).Their genes have recently been isolated and identified (S. COTECCHIA etal., 1988, Proc. Natl. Acad. Sci. USA, 85, 7159-7163; B. K. KOBILKA etal., 1987, Science, 238, 650-656; T. FRIELLE et al., 1987, Proc. Natl.Acad. Sci. USA 84, 7920-7924; L. J. EMORINE et al., 1987, Proc. Natl.Acad. Sci., USA, 84, 6995-6999; L. J. EMORINE et al., 1989, Science,245, 1118-1121). Analysis of these genes has enabled them to berecognized as belonging to a family of integral membrane receptorsdisplaying certain homologies (R. A. F. DIXON et al., 1988, AnnualReports in Medicinal Chemistry, 221-233; L. J. EMORINE et al., 1988,Proc. NATO Adv. Res. Workshop), in particular in respect of 7transmembrane regions which are coupled to regulatory proteins, known asG proteins, capable of binding guanosine triphosphate (GTP) molecules.

These membrane receptors, when they have bound the appropriate ligand(agonist or antagonist), undergo a change in conformation, which inducesan intra-cellular signal which modifies the behaviour of the targetcell.

In the case of β-adrenergic receptors, when they bind to catecholamineagonists, they catalyse the activation of a class of G proteins, whichin turn stimulates adenylate cyclase activity, while ARβ antagonists actin competition with the agonists for binding to the receptor and preventactivation of adenylate cyclase.

When adenylate cyclase is activated, it catalyses the production of anintracellular mediator or second messenger, in particular cyclic AMP.

The inventors have recently demonstrated new β-adrenergic receptors inman, designated Hu-ARβ₃, and in the mouse (International Application WO92/12,246), designated Mu-ARβ₃, and characterized by propertiesdifferent from those of the β₁ and β₂ receptors, in particular in thatthey behave differently with respect to substances which are,respectively, β₁ - and β₂ -receptor antagonists and agonists(International Application WO 90/08,775).

In particular, the Hu-ARβ₃ receptor consists, more especially, of asequence of 408 amino acids, and is considered to contain sevenhydrophobic transmembrane regions separated by intra- and extracellularhydrophilic loops, and the Mu-ARβ₃ receptor consists of a sequence of400 amino acids and also contains 7 transmembrane regions.

The previous studies relating to Hu-ARβ₃ and Mu-ARβ₃ showed, inparticular, that the β₃ -adrenergic receptor participates in disorderssuch as diabetes and/or obesity, in as much as it is expressed intissues which play an important part in metabolism (adipose tissues,skeletal muscles in particular).

Continuing his studies along these lines, one of the inventors sought todemonstrate such a β₃ -adrenergic receptor in cattle (Bo-ARβ₃), so as tobe able to have available a tool for regulating the amount of fats inthese animals, in particular with the object of improving the quality ofthe meat.

SUMMARY OF THE INVENTION

The subject of the present invention is a nucleotide sequence,characterized in that it corresponds to the cDNA of the bovine genecoding for the bovine β₃ -adrenergic receptor.

According to an advantageous embodiment of the said nucleotide sequence,it comprises the nucleotide sequence and the deduced amino acid sequence(SEQ ID No. 1) of formula (I).

In this sequence, the underlined ATG which occurs at position 107probably corresponds to the initiation codon for protein synthesis.

There is 85% homology between the bovine and human nucleotide sequencescoding for the β₃ -adrenergic receptor, and there is 76% homologybetween the bovine and murine nucleotide sequences coding for the β₃-adrenergic receptor.

The said sequence comprises, in particular, the following singlerestriction sites:

Bpu1102 I, Fok I, EcoR V, Bcg I, Nhe I, BspM I, Afl III, Age I, BstE II,BspH I, Bsg I, Nsp I, Nsp7524 I, NspC I, Sap I, BamH I, BstY I, Asc I,Sty I, Hinc II, Apa I, Bsp120 I, Bbe I, Ehe I, Kas I, Nar I, Ec1136 I,SaC I, Stu I, Fse I, Drd I, Tthlll I, Srf I, Bsu36 I, Sfc I, BstX I, AseI, Bsm I, Dra I.

The subject of the invention is also the fragments of the said sequencewhich are useful for expression of the corresponding peptide and/ordetection of the bovine gene coding for the bovine β₃ -adrenergicreceptor.

Among the said fragments, there may be mentioned:

the 78-base pair fragment which corresponds to nucleotides 218-295 ofthe sequence of formula I and which codes for the transmembrane regionTM1,

the 72-base pair fragment which corresponds to nucleotides 332-403 ofthe sequence of formula I and which codes for the transmembrane regionTM2,

the 66-base pair fragment which corresponds to nucleotides 434-499 ofthe sequence of formula I and which codes for the transmembrane regionTM3,

the 69-base pair fragment which corresponds to nucleotides 572-640 ofthe sequence of formula I and which codes for the transmembrane regionTM4,

the 72-base pair fragment which corresponds to nucleotides 713-784 ofthe sequence of formula I and which codes for the transmembrane regionTM5,

the 66-base pair fragment which corresponds to nucleotides 983-1048 ofthe sequence of formula I and which codes for the transmembrane regionTM6,

the 78-base pair fragment which corresponds to nucleotides 1070-1147 ofthe sequence of formula I and which codes for the transmembrane regionTM7.

The subject of the present invention is also cDNA clones, characterizedin that they comprise a sequence fragment coding for the bovine β₃receptor (Bo-ARβ₃).

According to the invention, the clone designated M13-6.6 comprises 2979base pairs, includes the sequence of formula I and comprises thefollowing single restriction sites: EcoR V, Bcg I, Nhe I, BstE II, BspHI, Bsg I, Sap I, BamH I, Asc I, Stu I, Fse I, Drd I, Srf I, Sfc I, AseI, Bsm I, Dra I, Bsp1407 I, Csp6 I, Rsa I, Ssp I, Dra III, Bgl II, AflII, Spe I, Tfi I, Hpa I, Nde I, EcoN I, BsaB I, Pvu I.

The subject of the present invention is also nucleotide probes,characterized in that they consist of a nucleotide sequence as isdefined above, or a fragment of the latter, labelled using a label suchas a radioactive isotope, a suitable enzyme or a fluorochrome.

The said nucleotide probes are characterized in that they hybridize withthe nucleotide sequences as are defined above but do not hybridize withthe genes coding for the β₁ - and β₂ -adrenergic receptors, or with themessenger RNA of the said β₁ - and β₂ -adrenergic receptors.

According to an advantageous embodiment of the said probe, its sequenceis homologous with or complementary to that of a segment of at least 10bp of the sequence I.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a and 1b depict restriction sites contained in cloned fragments.FIG. 1a depicts restriction sites contained in the 2,000 bp fragment.FIG. 1b depicts single restriction sites contained in the 3 kb fragment.

FIGS. 2.1 and 2.2 depict the homology among the bovine β₃ peptide (BETA3BOV; SEQ ID NO:2), the human β₃ peptide (BETA3 HU; SEQ ID NO:3), the ratβ₃ peptide (BETA3 RA; SEQ ID NO:4) and the murine β₃ peptide (BETA3 MO;SEQ ID NO:5). FIG. 2.1 depicts amino acids 1-240 of the peptides. FIG.2.2 depicts amino acids 241-405 of the bovine β₃ protein, amino acids241-408 of the human, and amino acids 241-400 of the rat and murineproteins.

FIGS. 3.1, 3.2, 3.3, and 3.4 depict the homology between the human β₃DNA (Huβ₃ ; SEQ ID NO:7) and the bovine β₃ DNA (bov β₃ ; SEQ ID NO:6).FIG. 3.1 depicts nucleotides 1-320. FIG. 3.2 depicts nucleotides321-640. FIG. 3.3 depicts nucleotides 641-960. FIG. 3.4 depictsnucleotides 961-1190.

FIG. 4 depicts a schematic of pRc/CMV-Boβ₃ -ADR.

FIG. 5 depicts a schematic of pRc/CMV, including a multisite linkercomprising the following single restriction sites; Hind III, BstX I, NotI, Xba I and Apa I.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

For the purpose of the present invention, "homologous sequence"encompasses not only sequences identical to the sequence I, or to afragment of the latter, but also those which differ therefrom only bythe substitution, deletion or addition of a small number of nucleotides,provided that the sequences thus modified have a specificity ofhybridization equivalent to that of the sequence (I) or of theunmodified segment in question.

Likewise, "complementary sequence" is understood to mean not onlysequences which are strictly complementary to the sequence (I) or to itssegments, but also modified sequences, as indicated above, possessing aspecificity of hybridization equivalent to that of the said strictlycomplementary sequences.

The hybridization conditions are defined as follows:

For the shortest probes, that is to say of approximately 10 toapproximately 100 nucleotides, suitable hybridization conditions are asfollows: 750 mM NaCl, 75 mM Tris-sodium citrate, 50 μg/ml salmon spermDNA, 50 mM sodium phosphate, 1 mM sodium pyrophosphate, 100 μM ATP, 10to 25% formamide, 1% Ficoll ("PHARMACIA", average molecular weight400.00), 1% polyvinylpyrrolidone, 1% bovine serum albumin, for 14 to 16h at 42° C.

For the longest probes, that is to say possessing more thanapproximately 100 nucleotides, suitable hybridization conditions arethose stated above for the shortest probes, but in which the mediumdefined above contains 40% of formamide instead of 10 to 25% offormamide.

According to an advantageous arrangement of this embodiment, the saidprobe can be advantageously defined by any one of the above nucleotidesequences, and in particular by the 2-kbase fragment which correspondsto the whole of the sequence of formula I.

The subject of the present invention is also a peptide and/or a peptidefragment, characterized in that it is encoded by a nucleotide sequenceas is defined above, and in that it displays β₃ -adrenergic receptoractivity.

β₃ -Adrenergic receptor activity is that defined in French PatentApplication No. 89/00,918, namely that, when the fragment is exposed atthe surface of a cell, it is capable of participating in the activationof adenylate cyclase in the presence of one of the following agonists:BRL 28410, BRL 37344, CGP 12177A, (1)-isoproterenol and carazolol; or,it is capable of being recognized by antibodies which do not recognizeeither the β₁ -adrenergic receptor or the β₂ -adrenergic receptor; or,it is capable of generating antibodies which do not recognize either theβ₁ receptor or the β₂ receptor.

According to an advantageous embodiment of the said peptide, itcomprises 405 amino acids and possesses the amino acid sequence (SEQ IDNo. 2) of formula II:

This peptide is designated hereinafter bovine β₃ -adrenergic receptor(Bo-ARβ₃).

The invention also comprises the peptides which are variants of thosedefined above, which contain certain mutations, without the peptideslosing the β₃ -adrenergic receptor properties.

Among these variants, there may be mentioned those which are recognizedby antibodies that recognize the transmembrane regions, as well as thosewhich are recognized by antibodies that recognize the regions other thanthe transmembrane regions.

The subject of the present invention is also fragments or combinationsof fragments of Bo-ARβ₃, according to the invention, and in particular:

a fragment of 26 amino acids corresponding to the segment 38-63 of theformula II and constituting the transmembrane region TM1,

a fragment of 24 amino acids corresponding to the segment 76-99 of theformula II and constituting the transmembrane region TM2,

a fragment of 22 amino acids corresponding to the segment 110-131 of theformula II and constituting the transmembrane region TM3,

a fragment of 23 amino acids corresponding to the segment 156-178 of theformula II and constituting the transmembrane region TM4,

a fragment of 24 amino acids corresponding to the segment 203-226 of theformula II and constituting the transmembrane region TM5,

a fragment of 22 amino acids corresponding to the segment 293-314 of theformula II and constituting the transmembrane region TM6,

a fragment of 26 amino acids corresponding to the segment 322-347 of theformula II and constituting the transmembrane region TM7.

The said fragments may advantageously be obtained by synthesis, inparticular by the Merrifield method.

The subject of the present invention is also a recombinant cloningand/or expression vector, characterized in that it comprises anucleotide sequence according to the invention.

For the purpose of the present invention, recombinant vector isunderstood to mean either a plasmid, a cosmid or a phage.

According to an advantageous embodiment of the said vector, it consistsof a suitable recombinant vector comprising, in particular, an origin ofreplication in a suitable host microorganism, in particular a bacteriumor a eukaryotic cell, at least one gene whose expression permitsselection either of the bacteria or of the eukaryotic cells which havereceived the said vector, and a suitable regulatory sequence, inparticular a promoter permitting expression of the genes in the saidbacteria or eukaryotic cells, into which vector is inserted a nucleotidesequence or a sequence fragment as are defined above, which vector is avector for the expression of a peptide, of a peptide fragment or of acombination of peptide fragments having bovine β₃ -adrenergic receptoractivity.

According to an advantageous arrangement of this embodiment, the saidvector consists of an expression vector pRc/CMV into which is inserted,in the multisite linker, at least the fragment coding for the bovine β₃-adrenergic receptor; such a plasmid has been designatedpRc/CMV-Boβ3-ADR, and has been deposited with the Collection Nationalede Cultures de Microorganismes [National Collection of MicroorganismCultures] (CNCM) held by the PASTEUR INSTITUTE, dated 15th Apr. 1993,under No. I-1297.

The subject of the present invention is also a suitable host cellobtained by genetic transformation, characterized in that it istransformed with an expression vector according to the invention.

Such a cell is capable of expressing a peptide of bovine origin havingβ₃ -adrenergic receptor acitivity.

According to an advantageous embodiment, the host cell consists, inparticular, of cells of the CHO (Chinese Hamster Ovary) line.

Another of the microorganisms used can consist of a bacterium, inparticular Escherichia coli.

It was not obvious that cattle have β₃ -adrenergic receptors whoseactivation unexpectedly enables the amount and quality of the fats to beregulated, thereby enabling the quality of bovine meat to be improved.

Advantageously, the bovine β₃ -adrenergic receptors according to theinvention constitute a tool for the selection of ligands participatingin the activation of these receptors, and make it possible to identifyand select β-adrenergic ligands which are specific for the β₃-adrenergic receptors, and especially ligands having more affinity andwhich are more selective for the bovine β₃ -adrenergic receptor than forthe human β₃ -adrenergic receptor.

According to the invention, the method for the selection andidentification of substances capable of behaving as a specific ligandwith respect to a peptide (bovine β₃ -adrenergic receptor) according tothe invention comprises:

bringing the said substance into contact with a host cell previouslytransformed with an expression vector as defined above, which host cellexpresses the said bovine peptide (bovine β₃ -adrenergic receptor), ifnecessary after suitable physical or chemical induction, and whichcontacting is carried out under conditions permitting the formation of abond between at least one of the specific sites and the said substanceif circumstances are appropriate, and

detecting the possible formation of a complex of the ligand-peptidetype.

Such a process makes it possible to select either ligands specific forthe β₃ -adrenergic receptor, or ligands specific for the bovine β₃-adrenergic receptor exclusively.

Besides the foregoing arrangements, the invention also comprises otherarrangements which will become apparent from the description whichfollows, reference being made to the attached drawings wherein:

It should, however, be clearly understood that these examples are givenonly by way of illustration of the subject of the invention, and in noway constitute a limitation thereof.

EXAMPLE 1

Isolation and Identification of the Bovine β₃ -Adrenergic Gene

Preparation of RNA

The bovine β₃ -adrenergic gene was isolated from a cDNA library of calfbrown adipose tissue, constructed in bacteriaphage λgtll.

To this end, the total RNA is extracted from calf brown adipose tissueby the guanidinium thiocyanate method, and the poly(A)⁺ messenger RNA isthen purified using oligo(dT) columns (Pharmacia Ref.: 27-9258-01).

The total RNA and messenger RNA were analysed by Northern blotting toverify the presence and size of the messengers of the desired gene.After electrophoresis, the RNA was transferred onto a positively chargednylon membrane (Amersham Hybond®-N+ ref. RPN 203B). This membrane isthen hybridized with a radiolabelled probe (radiolabelling: seescreening of recombinant phages, below), consisting of a 2900-base pairDNA fragment containing the whole of the murine β₃ -adrenergic genepreviously isolated in the laboratory (NAHMIAS et al., 1991, EMBO J.,10, 3721-3727; International Application WO 92/12,246). Afterhybridization with the radiolabelled probe, the filters are washed andexposed for several days to autoradiography film (KODAK X-OMAT AR); anapproximately 2.0-kilobase fragment is observed, both in the total RNAfraction and in the purified messenger RNA fraction. This confirms thatthe gene corresponding to the β₃ -adrenergic receptor is expressed incalf brown adipose tissue, and that the cDNA library can be constructedfrom this purified poly(A)⁺ messenger RNA.

To verify that the RNA source is indeed brown adipose tissue, theNorthern blot obtained above was hybridized with a radiolabelled probecorresponding to the gene for human uncoupling protein (hUCP). Thisprotein is only present in this type of adipose tissue, and may beregarded as a kind of "marker" for brown adipose tissue. With thisprobe, a strongly positive signal is detected.

Synthesis of cDNA

The corresponding cDNA is then synthesized, taking as template thepurified poly(A)⁺ messenger RNA and as primer for the synthesis of thefirst strand an oligo(dT)₁₅ primer originating from the "RiboClone cDNAsynthesis system" kit (Promega ref. C 2100). The synthesis of the firstcDNA strand takes place in the presence of AMV reverse transcriptase,and the synthesis of the second strand is carried out using two enzymesacting simultaneously (E. coli polymerase I and E. coli RNase H). Thedouble-stranded cDNA is then treated with T4 DNA polymerase so as toobtain blunt ends. The Promega C 2100 kit is used for all of thesereactions.

Next, adaptors containing EcoR I sites are added so as to be able toinsert the cDNA obtained into bacteriaphage λgtll under the followingconditions, described in the EcoR I Adaptor Ligation System I kit(Promega ref. C 1900):

the cDNA is centrifuged through a Sephacryl® S-400 matrix (kit) toremove small molecules; the adaptors are then added to the cDNA byligation in the presence of T4 DNA ligase, the mixture is left overnightand a second centrifugation is performed through a Sephacryl® S-400column so as to remove unbound adaptors.

Before inserting the cDNA thus treated into the λgtll vector, theadaptors are phosphorylated in the presence of T4 polynucleotide kinase.

Insertion of the cDNA into Bacteriaphage λgtll

The bacteriaphage λgtll used as vector originates from the "ProtocloneLambda gtll System" kit Promega ref. T 301/0-2). The DNA of the phage isdigested with EcoR I and dephosphorylated. Dephosphorylation preventsthe vector from closing up again.

Several ligations are performed with variable amounts of the cDNAobtained, with 0.5 μg of vector DNA, under the following conditions, foreach ligation: for 3 hours at room temperature in the presence of T4 DNAligase (Promega kit C1900).

An in vitro encapsidation is then performed using the "Packagene"extracts present in Promega kit T301/0-2.

After incubation at 22° C. for 2 hours, the reconstituted phageparticles are used to infect bacteria, in particular the strainY1090(r-) (Genotype: Δ(lacU169), proA+, Δ(lon), araD139, strA, supF,(trpC22::Tn10), (pMC9), hsd(r-, m+)), under the following conditions:the encapsidated phages are very greatly diluted (1/1,000 or 1/10,000);each dilution of phages is incubated with Y1090(r-) cells at 37° C. for30 minutes, and these infected bacteria are then plated out on anutrient medium (LB agar) contained in Petri dishes. The dishes areincubated overnight at 37° C., and the next day lytic plaques areobserved; each plaque corresponds to a recombinant phage. By countingthe number of lytic plaques and multiplying by the given dilutionfactor, the titer of the cDNA library is determined, and isapproximately 4 million recombinant phages. The background of the vectoralone without insert is 3.5%, which is entirely acceptable.

Screening of Recombinant Phages

On the basis of the results obtained, approximately 200,000 phages wereplated out on Petri dishes (LB agar medium) so as to be able to screenthem with a radiolabelled probe under the following conditions:

bacterial strain used: LE 392 (Genotype: F-, hsdR 574 (r-, m+), supE44,supF58, lacY1 or A(laclZY)6, galk2, galT22, metB1, trpR55, λ-);

probe: 2900-base pair DNA fragment (murine β₃ -adrenergic gene), asspecified above for Northern blotting, radiolabelled by random priming(Boehringer kit ref. 1004 760), incorporating 50 μCi of [α-³² P)dATP and50 μCi of [α-³² P]dCTP (Amersham references PB 10204 and PB 10205).

After transfer of the DNA from the lytic plaques onto Hybond®-N+membranes (Amersham ref. RPN 132B), the latter are hybridized with theradiolabelled probe, then washed and exposed overnight toautoradiography film.

17 hybridization signals were observed, 11 of which subsequently provedto be false positives. The 6 remaining clones (1, 3, 5, 6, 8 and 9) werepurified by four successive isolations, followed by a hybridization withthe murine β₃ -adrenergic probe described above.

Analysis of Positive Clones

To identify the clone(s) containing the entire bovine β₃ -adrenergicgene, that is to say the cDNA corresponding to the coding region for thewhole protein, 2 methods were used: amplification by PCR and cleavagewith a restriction endonuclease, with the object of finding among thepositive clones the one which contains the largest insert.

1) Amplification by PCR was carried out in lysate of phages(encapsidated phage particles) using the following two primers:

1218: 24-mer λgtll primer (sense strand) of formula: 5'd(GGTGGCGACGACTCCTGGAGCCCG)3' (SEQ. ID NO:8), and

1222: λgtll primer (antisense strand), also 24-mer, of formula: 5'd(TTGACACCAGACCAACTGGTAATG)3' (SEQ ID NO:9) (New England Biolabs).

In view of the fact that these primers hybridize on both sides of theinsertion site of the cDNA into the phage, it was possible in this wayto find out the size of the fragments inserted into the differentpositive clones.

2) The DNA of the 6 phages of interest was prepared and cut with therestriction enzyme EcoR I so as to verify the size of the inserts;hybridization with the murine β₃ -adrenergic probe enabled the clonecontaining the largest positive insert to be detected.

The outcome of these two approaches was that clone No. 6 was chosen fora more exhaustive analysis in view of the fact that it contains thelargest insert of desired cDNA (3 kilobases).

After cleavage of the phage λ with the restriction enzyme EcoR I, thefragment containing cDNA was inserted into bacteriophage M13tg131 so asto be able to sequence the gene.

EXAMPLE 2

Sequencing of the Bovine β₃ -Adrenergic Gene

The approximately 3-kb DNA fragment bounded by the EcoR I enzyme siteswas sequenced.

This DNA fragment was purified from the DNA of clone 6 and subclonedinto the EcoR I site of the vector M13tg131. The M13 clones which hadintegrated the DNA fragment in the 2 opposing orientations (6.3 and 6.6)were identified and sequenced.

To perform the sequencing reactions, the USB Sequenase Version 2.0 kit(United States Biochemical ref. 70770) was used.

The sequence was produced using specific primers, which hybridize withthe sense strand (clone M13-6.6) or with the antisense strand (cloneM13-6.3) according to the method of Sanger (MANIATIS et al., MolecularCloning, 2nd edition, pages 13.3-13.10).

The results obtained from the sequence of the 3-kilobase EcoR I fragmentshow the nucleotide sequence of bovine ARβ₃ (1215 bp) and non-codingregions (106 bp at the 5' end and 638 bp at the 3' end) (formula I). Therestriction sites contained in the 2,000-bp fragment are positioned onFIG. 1a (bov 6.6 short (pA)).

FIG. 1b shows the single restriction sites contained in the 3-kbfragment which was sequenced.

Comparison of the coding regions of the human and bovine β₃ genes (FIG.3) shows a strong homology (85% in respect of the nucleotide sequencesbetween bovine and human AR; comparison of the coding regions of thebovine and murine β₃ genes also shows strong homology (76% in respect ofthe nucleotide sequences between bovine AR and murine AR).

The bovine β₃ gene codes for the peptide of 405 amino acids whichdisplays a very large homology with the human β₃ peptide or the murineβ₃ peptide (FIG. 2), as indicated above.

EXAMPLE 3

Construction of a vector for the Expression of Bovine ARβ₃

The restriction map of the 2-kb fragment which was sequenced (FIG. 1a)shows the presence of a site of cleavage by the enzyme Srf I at position1598, that is to say 270 nucleotides upstream of the coding region ofthe bovine β₃ gene. DNA of the clone M13-6.6 was digested with theenzymes EcoR I and Srf I to liberate the 1598-base pair fragmentcontaining the coding region of the bovine β₃ gene and a portion of theuntranslated 3' region. This DNA fragment was purified and then insertedinto the expression vector pRc/CMV at the Hind III and Xba I cleavagesites (FIG. 4).

Since the ends generated by the enzymes Hind III and Xba I on the onehand, and EcoR I and Srf I on the other hand, are not compatible, carewas taken to treat the EcoR I and Srf I ends of the insert on the onehand with the Klenow fragment of polymerase I, and the Hind III and XbaI ends of the vector on the other hand with the Klenow fragment ofpolymerase I, so as to obtain blunt ends (MANIATIS et al., MolecularCloning, 2nd edition, pages 5.40-5.42).

The recombinant plasmid pRc/CMV-Boβ3-ADR shown in FIG. 5 was therebyobtained.

EXAMPLE 4

Pharmacological Properties of the Expression Product of the Bovine β₃Gene

a) Transfection of CHO-K1 Cells

To characterize better the bovine β₃ -adrenergic receptor, the bovine β₃gene is expressed at the surface of eukaryotic cells, which possess allthe elements needed for transduction of the signal.

The recombinant plasmid pRc/CMV of Example 3 was transfected into CHO-K1cells by a lypofectin transfection method; the transfected cells areselected with G418 (neomycin derivative).

More specifically, the said transfection method is carried out asfollows:

CHO-K1 cells (ATCC CCL 61) are cultured to confluence in a culturemedium containing; 45% DMEM medium, 45% F12-Ham medium, 10%heat-inactivated foetal calf serum, 2 mM glutamine, 100 U/ml penicillinand 100 μg/ml streptomycin.

1 μg of DNA of bovine β₃ plasmid pRc/CMV is mixed with 5 μl oflipofectin (Gibco) and 1,000 μl of the abovementioned culture mediumwithout serum.

This mixture is added to cells in culture, which are left to incubate at37° C. for 5 hours.

The medium is replaced with a culture medium, mentioned above,containing serum, and the cells are incubated again at 37° C. for 48hours. The cells are then distributed in 96 wells according to variabledilutions and incubated in the presence of geneticin (G418, Gibco) at aconcentration of 400 μg/ml in complete medium for approximately 10 days,the medium being changed every other day.

The different colonies obtained are then subcloned, first in 48 wellsand then in 6 wells, before screening.

Stable colonies are screened for their capacity to bind specifically to[¹²⁵ I]cyanopindolol and also for their capacity to stimulate adenylatecyclase in the presence of isoproterenol, in accordance with protocoldescribed in TATE et al., Eur. J. Biochem., 191, 196, 357-361.

Transfected cells stably expressing ARβ₃ bind [¹²⁵ I]cyanopindolol withan affinity equivalent to that of the corresponding ARβ₃ in man or inmouse and also obtained by cloning.

A set of subclones were selected from the stable clones; one of them,designated 62-26, was used for the pharmacological evaluation of bovineARβ₃.

b) Pharmacological Characteristics of the Bovine ARβ₃ Receptor

Pharmacological characterization of the bovine ARβ₃ receptor was carriedout using the stable clones 62-26. The pharmacological properties of 10β₃ -adrenergic ligands were determined by studies of stimulation ofadenylate cyclase and of binding of [¹²⁵ I]cyanopindolol.

Adenylate cyclase activation experiments were carried out according tothe protocol detailed in BLIN et al., Mol. Pharmacol., 1991, 44,1094-1104.

Briefly, preconfluent cells in six-well plates (0.6×10⁶ cells/well) areplaced in contact or otherwise with increasing doses of ligands for 30minutes at 37° C. Reaction is stopped by washing in PBS at 4° C. andadding 500 μl of 1N NaOH. After centrifugation and neutralization with1N acetic acid, the cell lysates are recovered and the total amount ofcAMP accumulated is determined using a commercial assay kit.

Study of the competitive binding of ligands was carried out on intactcells according to the protocol detailed in BLIN et al., Mol.Pharmacol., 1991, 44, 1094-1104. Briefly, 10⁵ cells are incubated with0.5 nM [¹²⁵ I]cyanopindolol in the presence or absence of increasingconcentrations of competitors for 30 minutes at 37° C. Reaction isstopped by dilution with ice-cold PBS, the cells are filtered off andthe radioactivity is measured in a gamma counter. The results wereanalysed using Graph-Pad© software.

Among the ligands tested, four are described as β₁ -, β₂ - and β₃-AR-receptor agonists: (-)-iso-proterenol, (-)-epinephrine,(-)-norepinephrine, BRL 37344; three are described as specific for theARβ₃ receptor (β₁ -, β₂ -AR antagonists): CGP12177A, ICI201651,bucindolol. Bupranolol was also tested since it is described as anantagonist of the three subtypes of receptor (BLIN et al., Br. J.Pharmacol., 1994, in press). Lastly, (-)-propranolol, described as apartial agonist of the human ARβ₃ receptor and antagonist of the mouseARβ₃ receptor (NAHMIAS et al., EMBO J., 1991, 10, 3721-3727), wastested.

The values of the adenylate cyclase activation constants (K_(act)), ofthe inhibition constants (K_(i)) and of the intrinsic activity (IA)corresponding to the ratio of the effect of the ligand at 10⁻⁴ M to theeffect of isoproterenol at 10⁻⁴ M, which are obtained for the differentligands, are presented in the table below. The four ligands which areagonists of the three subtypes of receptors (β₁ -, β₂ -, β₃ -AR) haveK_(act) and K_(i) values close to those obtained for the human ARβ₃receptor (BLIN et al., Br. J. Pharmacol., 1994, in press), mouse ARβ₃receptor (NAHMIAS et al., EMBO J., 1991, 10, 3721-3727) and rat ARβ₃receptor (GRANNEMAN et al., J. Pharmacol. Exp. Therap., 1991, 40,895-899; MUZZIN et al., J. Biol. Chem., 1991, 266, 24053-24058).

The specific ligands for the ARβ₃ receptor all have a smaller K_(act)value for the bovine ARβ₃ receptor compared to the human and mouse ARβ₃receptors, and hence improved efficacy in stimulating adenylate cyclase.(-)-Propranolol is a partial agonist at bovine ARβ₃, as for the humanARβ₃ receptor. In contrast, bupranolol, which is described as a potentantagonist for human and murine ARβ₃ receptors, is a partial agonist atthe bovine ARβ₃ receptor.

    __________________________________________________________________________             Mouse RAβ3-CHO                                                                             Human RAβ3-CHO                                                                             BovineRAβ3-CHO                       Binding                                                                              Accumul.                                                                           cAMP  Binding                                                                              Accumul.                                                                           cAMP  Binding                                                                              Accumul.                                                                           cAMP                 LIGANDS  Ki (nM)                                                                              Kact (nM)                                                                          IA    Ki (nM)                                                                              Kact (nM)                                                                          IA    Ki (nM)                                                                              Kact                                                                               IAM)                 __________________________________________________________________________    agonists                                                                      β1/β2/β3                                                       (-) isproterenol                                                                       --     99 ± 44                                                                         1.4 ± 0.1                                                                        620    4    0.9   84 ± 81                                                                           14 ± 2                                                                          0.9 ± 0.1         (- ) epinephrine                                                                       4,600 ± 1,850                                                                     23 ± 0.3                                                                        0.91 ± 0.03                                                                      20,650 ± 2.810                                                                    49 ± 5                                                                          1.00 ± 0.04                                                                      11,105 ± 7.345                                                                    50.7                                                                               0.8 ± 0.3         (-) norepinephrine                                                                     1,840 ± 600                                                                       13 ± 4                                                                          1.06 ± 0.06                                                                      475 ± 75                                                                          6.32 ± 0.7                                                                      1.00  423 ± 255                                                                         54 ± 4.3                                                                        1.00 ± 0.5        BRL 37344                                                                              290 ± 136                                                                         0.4 ± 0.1                                                                       1.07 ± 0.08                                                                      287 ± 92                                                                          15 ± 3                                                                          1.11 ± 0.12                                                                      2.13 ± 1.4                                                                        0.3                                                                                0.84 ± 0.1        β1/β2 antagonists/                                                  β3 agonists                                                              CGP 12177A                                                                             152 ± 19                                                                          41 ± 9                                                                          0.75 ± 0.08                                                                      88 ± 22                                                                           139 ± 44                                                                        0.68 ± 0.02                                                                      218 ± 161                                                                         1.41                                                                               0.93 ± 0.20       ICI 201651                                                                             239 ± 104                                                                         15 ± 1                                                                          1.02 ± 0.02                                                                      85 ± 12                                                                           20 ± 9                                                                          1.14 ± 0.14                                                                      27.7 ± 24                                                                         1.1                                                                                0.85 ± 0.1        Bucindolol                                                                             21 ± 5                                                                            40 ±14                                                                          1.11 ± 0.06                                                                      23 ± 10                                                                           7.0 ± 1.2                                                                       1.01 ± 0.10                                                                      73 ± 42                                                                           12.8                                                                               0.99 ± 0.10       partial agonist/                                                              antagonist                                                                    (- ) propranolol                                                                       150 ± 22                                                                          antagonist                                                                         --    145 ± 8                                                                           1,490 ± 550                                                                     0.51 ± 0.12                                                                      589 ± 74                                                                          661                                                                                0.71 ± 0.08                       406 ± 98                                                   antagonists                                                                   β1/β2/β3                                                       (-) bupranolol                                                                         42 ± 19                                                                           antagonist                                                                         --    50 ± 14                                                                           antagonist                                                                         --    85 ± 40                                                                           507                                                                                0.34 ± 0.01                       12 ± 1                                                     __________________________________________________________________________

As emerges from the foregoing, the invention is in no way limited tothose of its embodiments and modes of implementation and applicationwhich have just been described more explicitly; it encompasses, on thecontrary, all variants which may occur to the specialist in the field,without departure from the scope or range of the present invention.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 9                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2000 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 107..1321                                                       (D) OTHER INFORMATION: /function="BOVINE BETA-3 RECEPTOR"                     /product= "ADRENERGIC, BETA RECEPTOR"                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CCCAGGCCAGGGAAATCGCTCCCACGCCCCGATGCCCCCGCCGCTGAGCAGGGTGAGCTG60                GGAGACCCTTTCCCTCATTCCTTCCCGCCCCACGCGCGACGCGGGGATGGCTCCG115                    MetAlaPro                                                                     TGGCCTCCTGGGAACAGCTCTCTGACCCCGTGGCCAGATATCCCCACC163                           TrpProProGlyAsnSerSerLeuThrProTrpProAspIleProThr                              51015                                                                         CTGGCACCCAATACTGCCAACGCGAGTGGGCTGCCAGGGGTGCCCTGG211                           LeuAlaProAsnThrAlaAsnAlaSerGlyLeuProGlyValProTrp                              20253035                                                                      GCGGTGGCGCTGGCGGGGGCGCTGTTGGCGCTAGCGGTGCTGGCCACC259                           AlaValAlaLeuAlaGlyAlaLeuLeuAlaLeuAlaValLeuAlaThr                              404550                                                                        GTGGGAGGCAACCTGCTGGTAATCGTGGCCATCGCCCGGACGCCGAGA307                           ValGlyGlyAsnLeuLeuValIleValAlaIleAlaArgThrProArg                              556065                                                                        CTCCAGACCATGACCAACGTGTTCGTGACTTCGCTGGCCACAGCCGAC355                           LeuGlnThrMetThrAsnValPheValThrSerLeuAlaThrAlaAsp                              707580                                                                        CTGGTGGTGGGGCTCCTGGTCGTGCCCCCGGGGGCCACGTTGGCGCTG403                           LeuValValGlyLeuLeuValValProProGlyAlaThrLeuAlaLeu                              859095                                                                        ACCGGCCACTGGCCCCTGGGCGTCACCGGTTGCGAGCTGTGGACCTCA451                           ThrGlyHisTrpProLeuGlyValThrGlyCysGluLeuTrpThrSer                              100105110115                                                                  GTGGACGTGCTGTGTGTGACCGCCAGCATCGAAACCCTGTGCGCCCTG499                           ValAspValLeuCysValThrAlaSerIleGluThrLeuCysAlaLeu                              120125130                                                                     GCGGTGGACCGCTACCTGGCCGTGACCAACCCGCTGCGCTACGGCGCG547                           AlaValAspArgTyrLeuAlaValThrAsnProLeuArgTyrGlyAla                              135140145                                                                     CTGGTCACCAAACGCCGCGCCCTAGCAGCCGTGGTCCTGGTGTGGGTG595                           LeuValThrLysArgArgAlaLeuAlaAlaValValLeuValTrpVal                              150155160                                                                     GTGTCCGCCGCGGTGTCGTTTGCGCCCATCATGAGCAAATGGTGGCGC643                           ValSerAlaAlaValSerPheAlaProIleMetSerLysTrpTrpArg                              165170175                                                                     ATCGGGGCCGATGCCGAGGCGCAGCGTTGCCACTCCAACCCGCGCTGC691                           IleGlyAlaAspAlaGluAlaGlnArgCysHisSerAsnProArgCys                              180185190195                                                                  TGCACCTTCGCCTCCAACATGCCCTACGCGCTGCTCTCCTCCTCGGTC739                           CysThrPheAlaSerAsnMetProTyrAlaLeuLeuSerSerSerVal                              200205210                                                                     TCGTTCTATCTTCCGCTCCTGGTGATGCTCTTCGTCTACGCACGAGTT787                           SerPheTyrLeuProLeuLeuValMetLeuPheValTyrAlaArgVal                              215220225                                                                     TTCGTGGTGGCCACGCGCCAGCTGCGCTTGCTGCGCCGGGAGCTGGGT835                           PheValValAlaThrArgGlnLeuArgLeuLeuArgArgGluLeuGly                              230235240                                                                     CGCTTCCCGCCAGAGGAGTCTCCGCCGGCTCCTTCTCGCTCCGGATCC883                           ArgPheProProGluGluSerProProAlaProSerArgSerGlySer                              245250255                                                                     CCTGGCCTGGCGGGGCCGTGCGCCTCGCCCGCGGGGGTGCCCTCCTAC931                           ProGlyLeuAlaGlyProCysAlaSerProAlaGlyValProSerTyr                              260265270275                                                                  GGCCGGCGGCCGGCGCGCCTTCTGCCTCTGCGGGAACACCGCGCCCTG979                           GlyArgArgProAlaArgLeuLeuProLeuArgGluHisArgAlaLeu                              280285290                                                                     CGCACCTTGGGGCTCATCATGGGAACCTTCACTCTCTGCTGGTTGCCT1027                          ArgThrLeuGlyLeuIleMetGlyThrPheThrLeuCysTrpLeuPro                              295300305                                                                     TTCTTTGTGGTCAACGTGGTGCGCGCCCTCGGGGGCCCCTCTCTGGTG1075                          PhePheValValAsnValValArgAlaLeuGlyGlyProSerLeuVal                              310315320                                                                     TCCGGCCCCACTTTCCTCGCCCTTAACTGGCTGGGCTATGCCAACTCT1123                          SerGlyProThrPheLeuAlaLeuAsnTrpLeuGlyTyrAlaAsnSer                              325330335                                                                     GCCTTCAACCCGCTCATCTACTGCCGCAGCCCGGACTTTCGGAGCGCC1171                          AlaPheAsnProLeuIleTyrCysArgSerProAspPheArgSerAla                              340345350355                                                                  TTCCGCCGCCTGCTGTGTCGCTGCCGGCCGGAGGAGCACCTCGCCGCT1219                          PheArgArgLeuLeuCysArgCysArgProGluGluHisLeuAlaAla                              360365370                                                                     GCCTCCCCGCCCCGAGCCCCCTCCGGCGCCCCCACGGCCCTGACCAGC1267                          AlaSerProProArgAlaProSerGlyAlaProThrAlaLeuThrSer                              375380385                                                                     CCCGCTGGCCCCATGCAGCCCCCAGAGCTCGACGGGGCTTCCTGCGGA1315                          ProAlaGlyProMetGlnProProGluLeuAspGlyAlaSerCysGly                              390395400                                                                     CTTTCTTAGGCCTTGAAGAAACAACTCCATTGATCCGGAACCTTTGGAAAGCCTCT1371                  LeuSer                                                                        405                                                                           GGCCGGCCTCGGTTCAGAATGAGCCCCGTGGAGTTTCCCAGCTGGAAAACTCTGCCCTCC1431              CCAGCCTGACGACTGGGTCCTGGGAGGAGGCGCGGGGGCTGACTGGGGAGGGGAAATCCT1491              TACCAAGTGGGTTTTCGCTCTCTTTCTGAGAGAAGTTTTCTACACCCCAGCCCTGAACTT1551              CACCGCTGCCTCAGCAGCTCCCGCGTCTGGTTTCCCATGCCCAGGTGCCCGGGCAGGAGC1611              TGGGCTGCGTTTAGCCCCGGGACCCGCACCTGTCCCACTCGGGTGCTGTGTGCGCAGGGG1671              CAAGGCGGGCACCTTCATTCTGTTCCTTCTGCCGCCCAGACCCTGAGGAACCCACCGGGG1731              TGCTGGAGGCCCAGGCTGAGAAGAGGAAGGTGGGGAAGGTCACGGTTTGGGCTTCTGTCC1791              CTGGCTTCCTCACTGTAGACACACCTACCTCACAGCATTTTCAGGACTTTACTTTAGCCT1851              TTGGGGTGGGGGTGGGGGGGCGCTCCTGGTTTCCTGGGAAGGTGAACCATTAGAATGGGT1911              CCCTTTTCCTTTTGAAATCAAATTAATAAATGTTACTGAATGCAGTTTAAAAAAAAAAAA1971              AAAAAAAAAAAAAAAAAAAAAAAAAAAAA2000                                             (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 405 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetAlaProTrpProProGlyAsnSerSerLeuThrProTrpProAsp                              151015                                                                        IleProThrLeuAlaProAsnThrAlaAsnAlaSerGlyLeuProGly                              202530                                                                        ValProTrpAlaValAlaLeuAlaGlyAlaLeuLeuAlaLeuAlaVal                              354045                                                                        LeuAlaThrValGlyGlyAsnLeuLeuValIleValAlaIleAlaArg                              505560                                                                        ThrProArgLeuGlnThrMetThrAsnValPheValThrSerLeuAla                              65707580                                                                      ThrAlaAspLeuValValGlyLeuLeuValValProProGlyAlaThr                              859095                                                                        LeuAlaLeuThrGlyHisTrpProLeuGlyValThrGlyCysGluLeu                              100105110                                                                     TrpThrSerValAspValLeuCysValThrAlaSerIleGluThrLeu                              115120125                                                                     CysAlaLeuAlaValAspArgTyrLeuAlaValThrAsnProLeuArg                              130135140                                                                     TyrGlyAlaLeuValThrLysArgArgAlaLeuAlaAlaValValLeu                              145150155160                                                                  ValTrpValValSerAlaAlaValSerPheAlaProIleMetSerLys                              165170175                                                                     TrpTrpArgIleGlyAlaAspAlaGluAlaGlnArgCysHisSerAsn                              180185190                                                                     ProArgCysCysThrPheAlaSerAsnMetProTyrAlaLeuLeuSer                              195200205                                                                     SerSerValSerPheTyrLeuProLeuLeuValMetLeuPheValTyr                              210215220                                                                     AlaArgValPheValValAlaThrArgGlnLeuArgLeuLeuArgArg                              225230235240                                                                  GluLeuGlyArgPheProProGluGluSerProProAlaProSerArg                              245250255                                                                     SerGlySerProGlyLeuAlaGlyProCysAlaSerProAlaGlyVal                              260265270                                                                     ProSerTyrGlyArgArgProAlaArgLeuLeuProLeuArgGluHis                              275280285                                                                     ArgAlaLeuArgThrLeuGlyLeuIleMetGlyThrPheThrLeuCys                              290295300                                                                     TrpLeuProPhePheValValAsnValValArgAlaLeuGlyGlyPro                              305310315320                                                                  SerLeuValSerGlyProThrPheLeuAlaLeuAsnTrpLeuGlyTyr                              325330335                                                                     AlaAsnSerAlaPheAsnProLeuIleTyrCysArgSerProAspPhe                              340345350                                                                     ArgSerAlaPheArgArgLeuLeuCysArgCysArgProGluGluHis                              355360365                                                                     LeuAlaAlaAlaSerProProArgAlaProSerGlyAlaProThrAla                              370375380                                                                     LeuThrSerProAlaGlyProMetGlnProProGluLeuAspGlyAla                              385390395400                                                                  SerCysGlyLeuSer                                                               405                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 408 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       MetAlaProTrpProHisGluAsnSerSerLeuAlaProTrpProAsp                              151015                                                                        LeuProThrLeuAlaProAsnThrAlaAsnThrSerGlyLeuProGly                              202530                                                                        ValProTrpGluAlaAlaLeuAlaGlyAlaLeuLeuAlaLeuAlaVal                              354045                                                                        LeuAlaThrValGlyGlyAsnLeuLeuValIleValAlaIleAlaTrp                              505560                                                                        ThrProArgLeuGlnThrMetThrAsnValPheValThrSerLeuAla                              65707580                                                                      AlaAlaAspLeuValMetGlyLeuLeuValValProProAlaAlaThr                              859095                                                                        LeuAlaIleThrGlyHisTrpProLeuGlyAlaThrGlyCysGluLeu                              100105110                                                                     TrpThrSerValAspValLeuCysValThrAlaSerIleGluThrLeu                              115120125                                                                     CysAlaIleAlaValAspArgTyrLeuAlaValThrAsnProLeuArg                              130135140                                                                     TyrGlyAlaLeuValThrLysArgCysAlaArgThrAlaValValLeu                              145150155160                                                                  ValTrpValValSerAlaAlaValSerPheAlaProIleMetSerGln                              165170175                                                                     TrpTrpArgValGlyAlaAspAlaGluAlaGlnArgCysHisSerAsn                              180185190                                                                     ProArgCysCysAlaPheAlaSerAsnMetProTyrValLeuLeuSer                              195200205                                                                     SerSerValSerPheTyrLeuProLeuLeuValMetLeuPheValTyr                              210215220                                                                     AlaArgValPheValValAlaThrArgGlnLeuArgLeuLeuArgGly                              225230235240                                                                  GluLeuGlyArgPheProProGluGluSerProProAlaProSerArg                              245250255                                                                     SerLeuAlaProAlaProValGlyThrCysAlaProProGluGlyVal                              260265270                                                                     ProAlaCysGlyArgArgProAlaArgLeuLeuProLeuArgGluHis                              275280285                                                                     ArgAlaLeuCysThrLeuGlyLeuIleMetGlyThrPheThrLeuCys                              290295300                                                                     TrpLeuProPhePheLeuAlaAsnValIleArgAlaLeuGlyGlyPro                              305310315320                                                                  SerLeuValProGlyProAlaPheLeuAlaLeuAsnTrpLeuGlyTyr                              325330335                                                                     AlaAsnSerAlaPheAsnProLeuIleTyrCysArgSerProAspPhe                              340345350                                                                     ArgSerAlaPheArgArgLeuLeuCysArgCysGlyArgArgLeuPro                              355360365                                                                     ProGluProCysAlaAlaAlaArgProAlaLeuPheProSerGlyVal                              370375380                                                                     ProAlaAlaArgSerSerProAlaGlnProArgLeuCysGlnArgLeu                              385390395400                                                                  AspGlyAlaSerTrpGlyValSer                                                      405                                                                           (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 400 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetAlaProTrpProHisLysAsnGlySerLeuAlaPheTrpSerAsp                              151015                                                                        AlaProThrLeuAspProSerAlaAlaAsnThrSerGlyLeuProGly                              202530                                                                        ValProTrpAlaAlaAlaLeuAlaGlyAlaLeuLeuAlaLeuAlaThr                              354045                                                                        ValGlyGlyAsnLeuLeuValIleThrAlaIleAlaArgThrProArg                              505560                                                                        LeuGlnThrIleThrAsnValPheValThrSerLeuAlaThrAlaAsp                              65707580                                                                      LeuValValGlyLeuLeuValMetProProGlyAlaThrLeuAlaIle                              859095                                                                        ThrGlyHisTrpProLeuGlyAlaThrGlyCysGluLeuTrpThrSer                              100105110                                                                     ValAspValLeuCysValThrAlaSerIleGluThrLeuCysAlaLeu                              115120125                                                                     AlaValAspArgTyrLeuAlaValThrAsnProLeuArgTyrGlyThr                              130135140                                                                     LeuValThrLysArgArgAlaArgAlaAlaValValLeuValTrpIle                              145150155160                                                                  ValSerAlaThrValSerPheAlaProIleMetSerGlnTrpTrpArg                              165170175                                                                     ValGlyAlaAspAlaGluAlaGlnGluCysHisSerAsnProArgCys                              180185190                                                                     CysSerPheAlaSerAsnMetProTyrAlaLeuLeuSerSerSerVal                              195200205                                                                     SerPheTyrLeuProLeuLeuValMetLeuPheValTyrAlaArgVal                              210215220                                                                     PheValValAlaLysArgGlnArgArgLeuLeuArgArgGluLeuGly                              225230235240                                                                  ArgPheProProGluGluSerProArgSerProSerArgSerProSer                              245250255                                                                     ProAlaThrValGlyThrProThrAlaSerAspGlyValProSerCys                              260265270                                                                     GlyArgArgProAlaArgLeuLeuProLeuGlyGluHisArgAlaLeu                              275280285                                                                     ArgThrLeuGlyLeuIleMetGlyIlePheSerLeuCysTrpLeuPro                              290295300                                                                     PhePheLeuAlaAsnValIleArgAlaLeuValGlyProSerLeuVal                              305310315320                                                                  ProSerGlyValPheIleAlaLeuAsnTrpLeuGlyTyrAlaAsnSer                              325330335                                                                     AlaPheAsnProLeuIleTyrCysArgSerProAspPheArgAspAla                              340345350                                                                     PheArgArgLeuLeuCysSerTyrGlyGlyArgGlyProGluGluPro                              355360365                                                                     ArgValValThrPheProAlaSerProValAlaSerArgGlnAsnSer                              370375380                                                                     ProLeuAsnArgPheAspGlyTyrGluGlyGluArgProPheProThr                              385390395400                                                                  (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 400 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       MetAlaProTrpProHisArgAsnGlySerLeuAlaLeuTrpSerAsp                              151015                                                                        AlaProThrLeuAspProSerAlaAlaAsnThrSerGlyLeuProGly                              202530                                                                        ValProTrpAlaAlaAlaLeuAlaGlyAlaLeuLeuAlaLeuAlaThr                              354045                                                                        ValGlyGlyAsnLeuLeuValIleIleAlaIleAlaArgThrProArg                              505560                                                                        LeuGlnThrIleThrAsnValPheValThrSerLeuAlaAlaAlaAsp                              65707580                                                                      LeuValValGlyLeuLeuValMetProProGlyAlaThrLeuAlaLeu                              859095                                                                        ThrGlyHisTrpProLeuGlyGluThrGlyCysGluLeuTrpThrSer                              100105110                                                                     ValAspValLeuCysValThrAlaSerIleGluThrLeuCysAlaLeu                              115120125                                                                     AlaValAspArgTyrLeuAlaValThrAsnProLeuArgTyrGlyThr                              130135140                                                                     LeuValThrLysArgArgAlaArgAlaAlaValValLeuValTrpIle                              145150155160                                                                  ValSerAlaAlaValSerPheAlaProIleMetSerGlnTrpTrpArg                              165170175                                                                     ValGlyAlaAspAlaGluAlaGlnGluCysHisSerAsnProArgCys                              180185190                                                                     CysSerPheAlaSerAsnMetProTyrAlaLeuLeuSerSerSerVal                              195200205                                                                     SerPheTyrLeuProLeuLeuValMetLeuPheValTyrAlaArgVal                              210215220                                                                     PheValValAlaLysArgGlnArgHisLeuLeuArgArgGluLeuGly                              225230235240                                                                  ArgPheSerProGluGluSerProProSerProSerArgSerProSer                              245250255                                                                     ProAlaThrGlyGlyThrProAlaAlaProAspGlyValProProCys                              260265270                                                                     GlyArgArgProAlaArgLeuLeuProLeuArgGluHisArgAlaLeu                              275280285                                                                     ArgThrLeuGlyLeuIleMetGlyIlePheSerLeuCysTrpLeuPro                              290295300                                                                     PhePheLeuAlaAsnValLeuArgAlaLeuAlaGlyProSerLeuVal                              305310315320                                                                  ProSerGlyValPheIleAlaLeuAsnTrpLeuGlyTyrAlaAsnSer                              325330335                                                                     AlaPheAsnProValIleTyrCysArgSerProAspPheArgAspAla                              340345350                                                                     PheArgArgLeuLeuCysSerTyrGlyGlyArgGlyProGluGluPro                              355360365                                                                     ArgAlaValThrPheProAlaSerProValGluAlaArgGlnSerPro                              370375380                                                                     ProLeuAsnArgPheAspGlyTyrGluGlyAlaArgProPheProThr                              385390395400                                                                  (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1218 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       ATGGCTCCGTGGCCTCCTGGGAACAGCTCTCTGACCCCGTGGCCAGATATCCCCACCCTG60                GCACCCAATACTGCCAACGCGAGTGGGCTGCCAGGGGTGCCCTGGGCGGTGGCGCTGGCG120               GGGGCGCTGTTGGCGCTAGCGGTGCTGGCCACCGTGGGAGGCAACCTGCTGGTAATCGTG180               GCCATCGCCCGGACGCCGAGACTCCAGACCATGACCAACGTGTTCGTGACTTCGCTGGCC240               ACAGCCGACCTGGTGGTGGGGCTCCTGGTCGTGCCCCCGGGGGCCACGTTGGCGCTGACC300               GGCCACTGGCCCCTGGGCGTCACCGGTTGCGAGCTGTGGACCTCAGTGGACGTGCTGTGT360               GTGACCGCCAGCATCGAAACCCTGTGCGCCCTGGCGGTGGACCGCTACCTGGCCGTGACC420               AACCCGCTGCGCTACGGCGCGCTGGTCACCAAACGCCGCGCCCTAGCAGCCGTGGTCCTG480               GTGTGGGTGGTGTCCGCCGCGGTGTCGTTTGCGCCCATCATGAGCAAATGGTGGCGCATC540               GGGGCCGATGCCGAGGCGCAGCGTTGCCACTCCAACCCGCGCTGCTGCACCTTCGCCTCC600               AACATGCCCTACGCGCTGCTCTCCTCCTCGGTCTCGTTCTATCTTCCGCTCCTGGTGATG660               CTCTTCGTCTACGCACGAGTTTTCGTGGTGGCCACGCGCCAGCTGCGCTTGCTGCGCCGG720               GAGCTGGGTCGCTTCCCGCCAGAGGAGTCTCCGCCGGCTCCTTCTCGCTCCGGATCCCCT780               GGCCTGGCGGGGCCGTGCGCCTCGCCCGCGGGGGTGCCCTCCTACGGCCGGCGGCCGGCG840               CGCCTTCTGCCTCTGCGGGAACACCGCGCCCTGCGCACCTTGGGGCTCATCATGGGAACC900               TTCACTCTCTGCTGGTTGCCTTTCTTTGTGGTCAACGTGGTGCGCGCCCTCGGGGGCCCC960               TCTCTGGTGTCCGGCCCCACTTTCCTCGCCCTTAACTGGCTGGGCTATGCCAACTCTGCC1020              TTCAACCCGCTCATCTACTGCCGCAGCCCGGACTTTCGGAGCGCCTTCCGCCGCCTGCTG1080              TGTCGCTGCCGGCCGGAGGAGCACCTCGCCGCTGCCTCCCCGCCCCGAGCCCCCTCCGGC1140              GCCCCCACGGCCCTGACCAGCCCCGCTGGCCCCATGCAGCCCCCAGAGCTCGACGGGGCT1200              TCCTGCGGACTTTCTTAG1218                                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1227 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       ATGGCTCCGTGGCCTCACGAGAACAGCTCTCTTGCCCCATGGCCGGACCTCCCCACCCTG60                GCGCCCAATACCGCCAACACCAGTGGGCTGCCAGGGGTTCCGTGGGAGGCGGCCCTAGCC120               GGGGCCCTGCTGGCGCTGGCGGTGCTGGCCACCGTGGGAGGCAACCTGCTGGTCATCGTG180               GCCATCGCCTGGACTCCGAGACTCCAGACCATGACCAACGTGTTCGTGACTTCGCTGGCC240               GCAGCCGACCTGGTGATGGGACTCCTGGTGGTGCCGCCGGCGGCCACCTTGGCGCTGACT300               GGCCACTGGCCGTTGGGCGCCACTGGCTGCGAGCTGTGGACCTCGGTGGACGTGCTGTGT360               GTGACCGCCAGCATCGAAACCCTGTGCGCCCTGGCCGTGGACCGCTACCTGGCTGTGACC420               AACCCGCTGCGTTACGGGGCACTGGTCACCAAGCGCTGCGCCCGGACAGCTGTGGTCCTG480               GTGTGGGTCGTGTCGGCCGCGGTGTCGTTTGCGCCCATCATGAGCCAGTGGTGGCGCGTA540               GGGGCCGACGCCGAGGCGCAGCGCTGCCACTCCAACCCGCGCTGCTGTGCCTTCGCCTCC600               AACATGCCCTACGTGCTGCTGTCCTCCTCCGTCTCCTTCTACCTTCCTCTTCTCGTGATG660               CTCTTCGTCTACGCGCGGGTTTTCGTGGTGGCTACGCGCCAGCTGCGCTTGCTGCGCGGG720               GAGCTGGGCCGCTTTCCGCCCGAGGAGTCTCCGCCGGCGCCGTCGCGCTCTCTGGCCCCG780               GCCCCGGTGGGGACGTGCGCTCCGCCCGAAGGGGTGCCCGCCTGCGGCCGGCGGCCCGCG840               CGCCTCCTGCCTCTCCGGGAACACCGGGCCCTGTGCACCTTGGGTCTCATCATGGGCACC900               TTCACTCTCTGCTGGTTGCCCTTCTTTCTGGCCAACGTGCTGCGCGCCCTGGGGGGCCCC960               TCTCTAGTCCCGGGCCCGGCTTTCCTTGCCCTGAACTGGCTAGGTTATGCCAATTCTGCC1020              TTCAACCCGCTCATCTACTGCCGCAGCCCGGACTTTCGCAGCGCCTTCCGCCGTCTTCTG1080              TGCCGGTGCGGCCGTCGCCTGCCTCCGGAGCCCTGCGCCGCCGCCCGCCCGGCCCTCTTC1140              CCCTCGGGCGTTCCTGCGGCCCGGAGCAGCCCAGCGCAGCCCAGGCTTTGCCAACGGCTC1200              GACGGGGCTTCTTGGGGAGTTTCTTAG1227                                               (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "synthetic DNA primer"                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GGTGGCGACGACTCCTGGAGCCCG24                                                    (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "synthetic DNA primer"                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       TTGCACCCAGACCAACTGGTAATG24                                                    __________________________________________________________________________

We claim:
 1. An isolated and purified nucleotide sequence encoding abovine β3-adrenergic receptor comprising SEQ ID NO:1.
 2. A recombinantplasmid, comprising the nucleotide sequence according to claim
 1. 3. Therecombinant plasmid according to claim 2, further comprising an originof replication for replication in a host cell, at least one gene whoseexpression permits selection of said host cell transformed with saidplasmid, and a regulatory sequence, including a promoter permittingexpression of a protein having a bovine β3-adrenergic receptor activityin said host cell.
 4. The recombinant plasmid according to claim 3,wherein said plasmid is pRc/CMV-Boβ3-ADR and, wherein said plasmid isdeposited with the Collection Nationale de Cultures de Microorganismes(CNCM) held by the PASTEUR INSTITUTE, dated 15th Apr. 1993, under No.I-1297.
 5. A host cell transformed by a recombinant plasmid according toclaim
 3. 6. The transformed host cell according to claim 5, wherein saidhost cell is a CHO cell.
 7. The transformed host cell according to claim5, wherein said host cell is Escherichia coli.
 8. An isolated andpurified nucleic acid sequence consisting of a 72-base pair segmentwhich corresponds to nucleotides 332-403 of SEQ ID NO:1.
 9. An isolatedand purified nucleic acid sequence consisting of a 69-base pair segmentwhich corresponds to nucleotides 572-640 of SEQ ID NO:1.
 10. An isolatedand purified nucleic acid sequence consisting of a 66-base pair segmentwhich corresponds to nucleotides 983-1048 of SEQ ID NO:1.
 11. Anisolated and purified nucleic acid sequence consisting of a 78-base pairsegment which corresponds to nucleotides 1070-1177 of SEQ ID NO:1. 12.An isolated and purified protein, wherein the protein displaysβ3-adrenergic receptor activity and comprises SEQ ID NO:2.
 13. A methodfor detecting the binding of an agonist or antagonist to a proteinaccording to claim 12, comprising the steps of:contacting said agonistor antagonist with a host cell previously transformed by a vectorencoding said protein and wherein said host cell expresses said proteinon its surface, under conditions permitting binding between the proteinand said agonist or antagonist and detecting a complex formed betweensaid antagonist or agonist and said protein.